Project Summary/Abstract The incidence of new cancer diagnoses is declining each year, however the frequency of hepatocellular carcinoma (HCC) continues to rise, with a 5-year survival rate of 15%. HCC typically results from chronic liver disease and fibrosis, and liver transplant is the only curative treatment although recurrence can occur. Hence there is a need for improved therapies, and understanding how HCC develops and thrives in a fibrotic background will provide additional therapeutic targets. Wnt expression is upregulated in several cancers including HCC. Wnt ligands can function canonically to activate ?-catenin signaling, or noncanonically to regulate calcium signaling and antagonize ?-catenin. There are 19 total Wnts in the mammalian genome, and all hepatic cell types express Wnts which are secreted to cause autocrine or paracrine signaling. We and others have shown cell types including hepatocytes (HP) and macrophages (MP) produce different Wnts in several liver regeneration and injury models, however studies assessing cell-specific Wnt contributions in HCC are lacking. We hypothesize HP and MP produce Wnts important for carcinogenesis, and we developed cell-specific Wntless knockout animals (HP-KO and MP-KO). Wntless is required for Wnt secretion from a cell, therefore we functionally remove all Wnt activity. HP-KO and MP-KO and littermate controls will undergo a series of diethylnitrosamine and carbon tetrachloride injections (DEN/CCl4) to develop hepatic injury and eventual progression to fibrosis and HCC. This robust model leads to HCC by 5 months in 100% of mice. Furthermore, the DEN/CCl4 model allows us to assess HP and MP Wnts in the microenvironment and the tumor itself. Preliminary data suggests several Wnts are upregulated in control mice after DEN/CCl4. To test the role of HP- and MP-specific Wnts, we have developed two specific aims. Pilot studies identify MP-KO have greater tumor burden, inflammation, and injury than MP-CON at 5 months. Further, MP-KO exhibit frequent cholangiocarcinomas (CC) in addition to HCC. In Aim 1 we will test three hypotheses to explain increased tumorigenesis in MP-KO. Namely, (1) MP Wnts regulate macrophage polarization and reduce inflammation, (2) MP Wnts act to prevent combined HCC-CC (cHCC-CC), a more aggressive and lesser characterized form of liver cancer, or (3) MP Wnts are required for hepatic regeneration after injury, and blocking regeneration leads to a growth advantage for tumor cells. Furthermore, HP-KO and HP-CON have comparable injury and tumor burden at 5 months, however small CK19 positive nodules are present in HP-KO. CK19 is a marker of dedifferentiated tumors suggesting increased progenitor-like phenotype. We previously have shown HP secrete Wnt5a to terminate liver regeneration through ?-catenin inhibition after partial hepatectomy. In Aim 2 we will test the hypothesis that HP Wnts prevent tumor dedifferentiation through secretion of Wnt5a to inhibit ?-catenin activity, and we predict HP-KO will have fully dedifferentiated tumors by 7 months. Taken together, our data will elucidate Wnt signaling in HCC and may provide more targeted therapeutic options.